Revealing Structural Changes of Prion Protein during Conversion from

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Revealing structural changes of prion protein during conversion from #-helical monomer to #-oligomers by means of ESR and nanochannel encapsulation Che Yang, Wei-Lin Lo, Yun-Hsuan Kuo, Jason C Sang, Chung-Yu Lee, Yun-Wei Chiang, and Rita Pei-Yeh Chen ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/cb500765e • Publication Date (Web): 06 Nov 2014 Downloaded from http://pubs.acs.org on November 9, 2014

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ACS Chemical Biology

Revealing structural changes of prion protein during conversion from α-helical monomer to β-oligomers by means of ESR and nanochannel encapsulation

Che Yang1,2, Wei-Lin Lo1,2, Yun-Hsuan Kuo3, Jason C. Sang1,2, Chung-Yu Lee1,2, Yun-Wei Chiang3,*, and Rita P.-Y. Chen1,2,*

1

Institute of Biochemical Sciences, National Taiwan University, Taipei 10617, Taiwan 2

3

Institute of Biological Chemistry, Academia Sinica, Taipei 11529, Taiwan

Department of Chemistry, National Tsing Hua University, Hsinchu 30013, Taiwan

*To whom correspondence should be addressed:

1. Rita P.-Y. Chen, Institute of Biological Chemistry, Academia Sinica, No. 128, Sec. 2, Academia Rd., Nankang, Taipei, 11529, Taiwan; Tel.: +(886)-2-27855696; Fax: +(886)-2-2788-9759; E-mail: [email protected]

2. Yun-Wei Chiang, Department of Chemistry, National Tsing Hua University, No. 101, Sec. 2, Kuang-Fu Rd., Hsinchu, 30013, Taiwan; Tel.: +(886)-3-5715131, ext. 33345; E-mail: [email protected] 1

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Running head: β-oligomer structure of mouse prion protein

Key words: SDSL; DEER; misfolding; oligomer; amyloid; fibril

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Abstract Under non-denaturing neutral pH conditions, full-length mouse recombinant prion protein lacking the only disulfide bridge can spontaneously convert from an α-helical-dominant conformer (α-state) to a β-sheet-rich conformer (β-state), which then associates into β-oligomers, and the kinetics of this spontaneous conversion depends on the properties of the buffer used.

The molecular details of this structural

conversion have not been reported due to the difficulty of exploring big protein aggregates.

We introduced spin probes into different structural segments (three

helices and the loop between strand 1 and helix 1), and employed a combined approach of ESR spectroscopy and protein encapsulation in nanochannels to reveal local structural changes during the α-to-β transition.

Nanochannels provide an

environment in which prion protein molecules are isolated from each other, but the α-to-β transition can still occur.

By measuring dipolar interactions between spin

probes during the transition, we showed that helix 1 and helix 3 retained their helicity, while helix 2 unfolded to form an extended structure.

Moreover, our pulsed ESR

results allowed clear discrimination between the intra- and inter-molecular distances between spin labeled residues in helix 2 in the β-oligomers, making it possible to demonstrate that the unfolded helix 2 segment lies at the association interface of the β-oligomers to form cross-β structure.

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Introduction Prion protein (PrP) is an endogenous glycoprotein tethered to the outer membrane of cells, particularly neuronal cells, via a glycosylphosphatidylinositol anchor.

The structural conversion from the normal, mainly α-helical, cellular form

of prion protein (PrPC) to the β-sheet-rich, disease-causing form (PrPSc) is the cause of prion diseases, which are transmissible, fatal, neurodegenerative disorders.

The

structure of the infectious conformer PrPSc has not yet been solved and it is not clear which segments in prion protein form the cross-β structure in the resulting prion amyloid fibrils. fibrils (1-5).

Several structural models have been proposed for prion amyloid

The structures of three original α-helices in PrPC after the α-to-β

structural conversion are all different in these models.

For example, in the β-helical

trimer model, helix 1 is no longer an α-helix but involved in the formation of one left handed β-helix while the other two helices are retained (1); in the β-spiral model, the three original α-helices and 2 original β-strands are retained and two new β-strands (116-119 and 135-140) are formed (2); Cobb et al. (3) proposed a PrP fibril model in which seven new β-strands formed in the region of sequence 159-219 (covering helix 2 and 3) are stacked in a parallel, inregister manner.

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In 1999, Jackson et al. (6) reported that GdnHCl-denatured, DTT-reduced recombinant human PrP(91-231) dialyzed against a low pH buffer can refold to a monomeric β-sheet-rich conformer, named β-PrP, which shows partial protease resistance and is prone to associating into soluble oligomers (β-oligomers) or fibrils at high ionic strength.

The pH is a key factor in determining whether β-oligomers or

amyloid fibrils are formed (7). same monomer structure.

It is unclear whether oligomers and fibrils share the

Very recently, we reported that the disulfide bond between

C179 and C214 is important in maintaining the native structure of full-length recombinant mouse PrP, because, in the absence of this bond, full-length PrP converts spontaneously and slowly (in days) to β-sheet-rich oligomers when dissolved in 0.5 mM NaOAc (pH 7) in the absence of a denaturant (8).

The β-oligomers formed in

this way also showed cytotoxicity for neuroblastoma cells (9).

However, the

determination of the molecular details of the spontaneous conversion of prion protein to β-form monomers and oligomers has not been possible using existing biophysical tools.

ESR has been shown to be useful for studying prion protein (10-13) and the amyloid fibrils of prion peptides (3, 14, 15).

In this study, in addition to circular

dichroism (CD) spectroscopy, analytical ultracentrifugation (AUC), and electron

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microscopy (EM), we used a combined approach of electron spin resonance (ESR) and protein incorporation in nanochannels to unveil the molecular details of the structural conversion of recombinant mouse prion protein mPrP(23-230) from an α-helical monomeric conformer (the α-state) to a β-oligomeric conformer (the β-state).

Two spins were introduced to each α-helix individually and the spin-spin

distance in monomeric PrP or in oligomeric PrP could provide information to address the question whether this α-helix remains the α-helical structure after structural conversion. Wild-type mPrP has only two Cys residues (C179 and C214), which form a disulfide bridge.

In order to label spin on prion protein, the mutant

mPrP-CtoA, lacking the disulfide bridge, was constructed by replacing these two Cys residues in mPrP(23-230) with Ala by site-directed mutagenesis.

Several variants of

mPrP-CtoA in which either one or two residues in different structural segments (three α-helices and the loop between strand 1 and helix 1) were replaced by Cys were prepared for a spin-labeling study (Figure 1a) by reacting a methane thiosulfonate spin probe, MTSSL, with the sulfhydryl group of Cys to introduce a nitroxide side-chain, designated as R1.

The buffer conditions for these spin-labeled proteins

to adopt an α-state or β-state conformer were carefully examined by CD spectroscopy. To be consistent with the sample condition in ESR, proteins for CD measurement were also encapsulated in nanochannels (Figure 1b) in this study.

Oligomerization

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was verified by AUC, oligomer morphology was observed using EM, and structural changes in spin-labeled segments during the structural conversion were explored by continuous wave (cw)-ESR or pulsed ESR.

Results and Discussion A combined ESR and nanochannel approach has recently been shown to be useful in biophysical studies (15-24).

In solution, the ESR lineshape of a

spin-labeled protein is commonly dominated by unwanted effects, such as the tumbling motion of the protein and side-chain internal motion.

A conventional way

to reduce the unwanted effects is to increase the viscosity of the buffer by adding viscosity agents, such as glycerol or sucrose; however, we found that addition of glycerol or sucrose greatly increased speed of oligomerization and hence led to the difficulty in recording the ESR spectrum of protein in α-state.

Encapsulation of the

spin-labeled proteins in nanochannels substantially reduces protein tumbling motion and side-chain internal motion due to nanoconfinement (15, 25-27).

As a result, the

ESR lineshape becomes more sensitive to backbone dynamics, making it easier to discriminate between different secondary structures and local changes in a protein. As the use of ESR and protein encapsulation in nanochannels allows measurement in the absence of viscosity-altering agents, this approach was used throughout this study.

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The disulfide-lacking prion protein exhibits a native-like tertiary contact in the α-state, but not the β-state To examine whether the α-state of the disulfide-lacking prion protein used in this study had a similar tertiary structure to wild-type mouse PrP, a mutant in which S132 and Q217 were replaced with Cys was constructed and reacted with MTSSL to generate the double spin-labeled protein S132R1/Q217R1.

S132 and Q217 are far

apart in the primary structure but close in the tertiary structure of the mouse prion protein.

Maiti and Surewicz (28) reported that a low pH helps to solubilize

disulfide-reduced or Cys-free human prion proteins and prevent them forming insoluble aggregates. or

β-state

was

For CD and cw-ESR measurement, S132R1/Q217R1 in the αencapsulated

in

SBA-15

nanochannels

(see

Methods).

S132R1/Q217R1 was dissolved in buffers at a protein concentration of 10 mg/mL, encapsulated in nanochannels, and the CD spectra were recorded.

When

S132R1/Q217R1 was dissolved in 0.5 mM NaOAc (pH 5), two negative peaks at 208 and 220 nm in its CD spectrum suggested an α-helical structure (Figure 2a). S132R1/Q217R1 solution (10 mg/mL) was diluted in the same buffer to 0.1 mg/mL for running AUC and the data showed that it adopted a monomeric structure (Figure 2c).

Monomeric S132R1/Q217R1 could not be seen by TEM due to resolution

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limitations of TEM (Figure 2e).

On the other hand, when the protein was dissolved

in 10 mM NaOAc (pH 5), the negative peak at around 216 nm in its CD spectrum suggested that the protein adopted a β-rich structure (Figure 2b) and the AUC data clearly showed the appearance of oligomers (~11-mer) (Figure 2d). oligomer morphology was seen in its TEM image (Figure 2f).

Moreover,

To sum up, CD, AUC,

and TEM results (Figure 2a-f) showed evidence that S132R1/Q217R1 is in the α-state in 0.5 mM NaOAc (pH 5) and β-state in 10 mM NaOAc (pH 5).

To obtain cw-ESR

spectra, S132R1/Q217R1 in the α- or β-state was encapsulated in SBA-15 nanochannels.

The normalized cw-ESR spectra for the double-labeled mutant

S132R1/Q217R1 and the sum of the normalized spectra for the single-labeled mutants S132R1 and Q217R1 (S132R1+Q217R1) in the α-state are shown in Figure 2g and those in the β-state in Figure 2h.

The spectra were normalized to the same double

integration, so a spectrum of lower magnitude indicates greater linewidth broadening. Clear linewidth broadening was seen on comparing S132R1/Q217R1 to S132R1+Q217R1 in the α-state (Figure 2g), but not in the β-state (Figure 2h).

This

suggests that the intra-molecular spin-spin distance in the α-state mutant is within the cw-ESR-sensitive distance range (ca. 0.8 ~ 2 nm) and, therefore, the corresponding distance distribution P(r) can be obtained using the Tikhonov-based deconvolution method (29) and is shown in Figure 2i.

The major distance population of 1.16 nm is

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consistent with the distance between S132 and Q217 measured based on the known structure of mouse prion protein (Figure 2j), indicating that the disulfide-lacking mutant protein has a similar tertiary structure to wild-type PrP.

Examination of changes in helices 1, 2, and 3 during β-oligomer formation D144 and R151 in helix 1, N174 and N181 in helix 2, and Q212 and Q217 in helix 3 were selected for spin-labeling in order to examine changes in the helicity of these three α-helices in the α- and β-states.

The inter-residue distances in the three

prion mutants D144/R151, N174/N181, and Q212/Q217 are about, respectively, 1.08 nm (2 turns away), 1.08 nm (2 turns away), and 1.27 nm (1.5 turns away and on the opposite site of the helix) (Figure 1a).

The double spin-labeled mutant proteins

D144R1/R151R1, N174R1/N181R1, and Q212R1/Q217R1 were therefore prepared for ESR, CD, AUC, and TEM studies.

The structure of helix 1 was studied using D144R1/R151R1 in the α- and β-states; note that the samples were encapsulated in nanochannels prior to the CD and ESR studies, but not in the AUC and TEM studies.

As Figure 2a-f, Figure 3a-f

showed evidence that D144R1/R151R1 is in the α-state in 0.5 mM NaOAc (pH 5) and β-state in 5 mM NaOAc (pH 7).

An inter-spin dipolar interaction was seen in

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both states, as the spectra for D144R1/R151R1 were clearly broader in linewidth than the summed spectra for the corresponding single-labeled proteins (D144R1+R151R1) (Figure 3g and h).

The results of the deconvolution analysis (distance distribution;

P(r)) showed that the intermolecular spin-spin distances for D144R1/R151R1 were 0.99 nm in the α-state (Figure 3i) and 1.01 nm in the β-state (Figure 3j), values close to one other and consistent with a distance of 1.08 nm expected for a local structure comprised of two α-helical turns (0.54 nm/turn x 2 turns), suggesting that helix 1 retains its α-helical conformation after β-oligomer formation.

The local structure of helix 2 was studied using N174R1/N181R1 in the α- and β-states.

Figure 4a-f showed evidence that N174R1/N181R1 is in the α-state in 0.5

mM NaOAc (pH 5) and β-state in 0.5 mM NaOAc (pH 7).

The cw-ESR results for

N174R1/N181R1 showed that spectral broadening was seen in the α-state (Figure 4g), but not the β-state (Figure 4h).

The deconvolution of the ESR spectrum of

N174R1/N181R1 in the α-state yielded an intramolecular spin-spin distance of 1.3 nm (Figure 4i), close to the distance of two α-helical turns.

Taken together, the

results indicated that helix 2 retained its helical structure in the α-state, but not the β-state, suggesting that unfolding of helix 2 occurs during the α-to-β structural conversion.

As the inter-spin distance of N174R1/N181R1 in the β-state could not

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be determined due to the limitation of cw-ESR, a pulsed double electron-electron resonance (DEER)-ESR study, which allows measurement of distance within the range 1.5 to 6 nm, was performed.

The main panel in Figure 4j shows the

baseline-subtracted time-domain signals obtained by pulsed ESR and the inset shows the distance distribution P(r) determined by Tikhonov analysis (30).

Note that the

DEER measurement was performed in the absence of nanochannels.

The P(r)

clearly showed two distance populations at 2.4 nm and 3.1 nm; the distance of 2.4 nm is probably the intra-molecular spin-spin distance between N174R1 and N181R1, as it is consistent with the estimated distance between N174 and N181 in an extended β-strand (0.35 × 7 = 2.45 nm), so the distance of 3.1 nm was therefore assigned to the inter-molecular spin-spin distance between adjacent prion proteins (between N174R1 in molecule(i) and N181R1 in molecule(i+1) in a β-oligomer.

These data demonstrate

that helix 2 is unfolded during the formation of β-PrP and is situated in the β-PrP association interface during the process of β-oligomer formation.

In cw-ESR spectra

of 7 single spin-labeled mutant proteins in the α- or β-state, only N174R1 and N181R1 β-oligomers showing spin-spin interaction also supports this conclusion (Supplementary Figure S1).

The strucutre of helix 3 was studied using Q212R1/Q217R1 in the α- and

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β-states.

Figure 5a-f. showed evidence that Q212R1/Q217R1 is in the α-state in 0.5

mM NaOAc (pH 5) and β-state in 10 mM NaOAc (pH 7).

Like D144R1/R151R1,

dipolar spectral broadening was clearly observed in both the α-state (Figure 5g) and β-state (Figure 5h).

Deconvolution analysis gave inter-spin distances for

Q212R1/Q217R1 in the α- and β-state of 1.23 and 1.27 nm, respectively, close to the distance between side-chains of Q212 and Q217 (1.2~1.5 nm) obtained from the nuclear magnetic resonance (NMR) structure of mouse prion protein (PDB: 1AG2), suggesting that helix 3 retains its helicity during the structural conversion (Figure 5i&j).

Direct observation of spontaneous structural conversion from α-PrP monomer to β-PrP monomer Previously, we reported that, in 0.5 mM NaOAc, pH 7, the disulfide-lacking full-length recombinant mouse PrP can convert from its native-like α-helical structure (α-PrP) to a partially unfolded conformer (β-PrP) and that the β-PrP monomer is unstable and tends to form β-oligomers (8).

Since residue 174 had been shown to be

at the association interface of β-oligomers (Figure 4), a single-labeled mutant N174R1 in the α-state was prepared.

β-PrP association could be revealed by linewidth

broading in the cw-ESR spectrum due to spin-spin interaction in the association

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interface of oligomers. By incubating N174R1 α-PrP in solution or in nanochannels for 60 days, we demonstrated that the monomeric β-PrP formed in nanochannels was stable and did not associate with other β-PrP molecules inside the nanochannels (Supplementary Figure S2).

The CD spectra of N174R1 incubated in solution

clearly showed a conversion from the α-state to the β-state over a 60-day period (Supplementary Figure S2a), whereas the ESR spectra of N174R1 incubated in nanochannels displayed no sign of intermolecular dipolar broadening (Supplementary Figure S2b), showing that N174R1 did not associate with other encapsulated prion molecules in nanochannels.

We then performed a time-course ESR analysis of the

encapsulated S132R1/Q217R1 and N174R1/N181R1 to monitor how the local environment of the spins varied with time during the transition from monomeric α-PrP to monomeric β-PrP.

S132R1/Q217R1 and N174R1/N181R1 were dissolved

in 0.5 mM NaOAc (pH 5) at a concentration of 10 mg/mL.

An aliquot was taken out,

encapsulated in nanochannels, and confirmed to be in the α-state by recording their CD spectra immediately (marked as “0 day”). The concentrated protein solutions were incubated at 4 ℃.

At the 7th or 22th day, an aliquot was taken out,

encapsulated in nanochannels, and the CD spectra were recorded. The CD spectra of S132R1/Q217R1 (Figure 6a) and N174R1/N181R1 (Figure 6b) showed that these protein in buffer spontaneously turned into β-oligomers over the incubation period

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examined.

For ESR studies, S132R1/Q217R1 and N174R1/N181R1 in 0.5 mM

NaOAc (pH 5) (10 mg/mL) were encapsulated in nanochannels immediately.

The

proteins trapped in the nanochannels were incubated at 4 ℃ for the indicated time, and the ESR spectra recorded.

The normalized cw-ESR spectra of encapsulated

S132R1/Q217R1 over time (Figure 6c) showed that the peak magnitude increased gradually with incubation time, indicating a gradual decrease in inter-spin dipolar interactions. This observation confirmed that residue-132 and residue-217 were torn apart when the native-like structure of S132R1/Q217R1 was partially unfolded in the β-PrP monomer.

A linewidth decrease was also observed for α-state

N174R1/N181R1 after incubation in nanochannels for 5 days, indicating that helix 2 was unfolded during the structural transition from α-PrP to β-PrP monomer (compare the black and red lines in Figure 6d).

As also shown in Figure 6d, β-state

N174R1/N181R1 was formed from the association of N174R1/N181R1 β-PrP monomer in solution, the magnitude of its normalized cw-ESR spectrum (blue line) was reduced (due to the inter-molecular dipolar interactions in oligomers) compared to that after 5 days of incubation in nanochannels (red), consistent with helix 2 being at the association interface during oligomerization.

In this study, we report the local structure of three α-helices during the

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conversion process from α-helical monomer to β-oligomers, rather than to amyloid fibrils, as examined in most other studies mentioned in the introduction section (1-5). Like in vitro prepared amyloid fibrils, our β-oligomers were not infectious, as mice injected intracerebrally with our β-oligomers did not show any clinical symptoms during the first passage (Supplementary Figure S3).

Do the β-oligomers share the

same cross-β structure with the in vitro prepared amyloid fibrils?

Using denaturant

titration and NMR spectroscopy, Hosszu et al. (31) have reported that helices 1 and 3, but not helix 2, retained some helical character in acid-induced β-PrP formed from human PrP(91-231).

Again using NMR, Bjorndahl et al. (32) reported that the

“NNQNNF” region (hamster PrP sequence 170-175) might be involved in the initial helix-to-β conversion process during acid-induced β-PrP formation.

Our

β-oligomers prepared in neutral and non-denaturing conditions have different structural properties to those β-oligomers prepared under acidic and reduced conditions in the presence of denaturant. The differences are that i) our β-oligomers were as proteinase K-sensitive as wildtype mouse PrP (Supplementary Figure S4), while acid/denaturant-induced β-oligomers showed slight resistance to proteinase K digestion (6, 32); and ii) the formation of our β-oligomers is irreversible (8, 32), while formation of the acid/denaturant-induced β-oligomers is reversible at a pH higher than 1.7 (8, 32).

Nevertheless, our finding that helix 2 is the site where the α-to-β

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structural transition of β-PrP formation takes place is consistent with the results of the two groups above (31, 32).

We provide clear evidence that, during the α-to-β

conversion process, the helix 2 of prion protein is unfolded to an extended β-strand while the helicity of the other two helices remained the same and that the extended strand formed by unfolding of helix 2 associate with the same strand of another PrP molecule to form cross-β structure in the β-oligomers.

This structure is complete

different from the previously proposed fibril structures (1-5), suggesting that the structure of prion β-oligomers which are produced in a non-denaturing condition is different from the structures of prion amyloid fibrils which are prepared in a partially denatured condition.

One important finding of our study is that, using a combined ESR and nanochannel encapsulation approach, the α- and β-states, as well as the transition process, can be conveniently monitored so as to reveal dynamical/structural changes in different structural segments and local environments.

Smythe et al. have argued

that flexibility of the spin-labels employed in the ESR studies can affect the accuracy of conformational identification (33).

Indeed MTSSL labeled “R1” side-chain has a

length of ~0.5 nm and five rotatable bonds which inevitably contributes some uncertainty to distance distribution.

However, nanochannel encapsulation can

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reduce disordering of R1 side-chain and hence reduce the ambiguity in distance measurement.

In the previous study, a 26-residue prion protein peptide was doubly

spin-labeled and investigated in two comparative conditions, bulk solvent versus nanochannel (27).

By varying buffer condition, secondary structure of the peptide

can change between helix and β-hairpin (25).

In bulk solvent the distance

distribution within the peptide changes little between the two buffer-induced secondary structures.

The structural difference (< 0.5 nm) between the helix and

β-hairpin structures was compromised by the disordering of the R1 side chain in bulk solvent. Whereas, upon encapsulation of the peptide into nanochannels, it was shown that the disordering of the R1 side chain is substantially reduced, rendering the differences in the two secondary structures more clearly revealed in the distance distributions by cw-ESR.

The findings proved that the combined approach of

cw-ESR and nanochannel is useful for distinguishing local structural difference.

In

the present study, the same approach and tool were employed to investigate the change in the secondary structure of a full-length prion protein during the spontaneous structural conversion. In the nanochannels, hardly any change was found in the ESR spectra (cf. Figures 3 and 5) corresponding to the helix 1 and helix 3 between the α and the β states, while the CD spectra clearly showed the α-to-β conversion of the whole protein. Only the study of helix 2 displayed substantial spectral difference

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between the two states. The observed spectral changes are clearly correlated with structural segments. Therefore, the local structural changes in helix 2 are unambiguously identified using this combined approach of cw-ESR and nanochannel. This combined approach made it possible to determine the molecular interactions involved in the structural changes within an individual protein molecule and those between different molecules.

Methods Protein expression and purification.

The mouse PrP gene cloned in the

pET101/D-TOPO vector was kindly provided by Dr Ilia V. Baskakov (Center for Biomedical Engineering and Technology, University of Maryland Biotechnology Institute, USA); the expressed prion protein contains mouse PrP residues 23-230 with an additional Met at the N-terminus.

Mutations S132C, D144C, R151C, N174C,

N181C, Q212C, Q217C, S132C/Q217C, D144C/R151C, N174C/N181C, and Q212C/Q217C were constructed by site-directed mutagenesis. The proteins were expressed in BL21 Star™ (DE3) cells (Invitrogen) and were purified using a protocol modified from a published method (34).

An overnight culture was used to inoculate

fresh TB medium containing ampicillin (100 µg/mL) at a ratio of 2:100 and the cells were grown at 37 ℃ with vigorous shaking (250 rpm) for 3 h (A600 nm = ~0.6), then

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protein expression was induced by adding IPTG at a final concentration of 1 mM and incubation was continued for 5 h, then the cells were harvested by centrifugation at 1,900 g at 4 ℃ for 10 min.

The cell pellet (about 34 g from 2.4 L of culture) was

resuspended in 300 mL of cell lysis buffer (50 mM Tris-HCl, 100 mM NaCl, pH 8.0) and the cells lysed by addition of 0.5 X CelLyticB (Sigma), 0.15 mg/mL of lysozyme, 25 µg/mL of DNase I, 7 mM MgCl2, and 1 mM PMSF.

The lysate was centrifuged

at 6,000 g at 4 ℃ for 30 min and the pellet resuspended in freshly prepared IMAC A buffer (8 M urea, 100 mM Na2HPO4, 10 mM Tris-HCl, 2 mM TCEP, pH 8.0) and the suspension incubated at room temperature for 2 h, then centrifuged at 18,000 g at 4 ℃ for 20 min.

The supernatant was applied at a flow rate of 150 cm/h to a column

packed with chelating Fast Flow Sepharose resin (GE Healthcare) precharged with Ni-ions, which was then washed with 5 column volumes of IMAC A buffer and prion protein eluted with 4 column volumes of IMAC A buffer containing 20 mM EDTA and further purified by reversed-phase chromatography on a C5 HPLC column (Discovery BIO Wide Pore C5, 10 µm, 25 cm x 10.0 mm, Supelco, Sigma-Aldrich) using a linear gradient of 29-47 % buffer B in 30 min (buffer A: 94.9 % water, 5 % acetonitrile, 0.1 % TFA; buffer B: 99.9 % acetonitrile, 0.1 % TFA) at a flow rate of 3 mL/min. The proteins were identified by SDS-PAGE and ESI-TOF mass spectrometry, lyophilized, and stored at -30 ℃.

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Spin-labeling.

In order to carry out ESR studies, mutant proteins were labeled with

(1-oxyl-2,2,5,5-tetramethylpyrroline-3-methyl) (MTSSL) (Alexis Biochemicals).

methanethiosulfonate

spin

label

Four milligrams of the PrP mutant protein was

dissolved in 400 µL of DMSO on ice, then 50 mM MOPS (pH 6.5) buffer was added to give a final volume of about 974 µL.

The protein solution was then mixed with

20 µL of 50 mM TCEP, then 6 µL of 126 mM MTSSL was added and the solution incubated overnight at room temperature in the dark. The molar ratio of cysteine to MTSSL was 1:5.

Reversed-phase HPLC was used to remove excess free MTSSL

and unlabeled protein.

Circular dichroism (CD) spectroscopy.

Protein samples were dissolved in the

indicated buffer to prepare the proteins in the α-state or β-state at a high protein concentration (10 mg/mL)

To obtain CD spectra in nanochannels, a previously

reported procedure was followed (24).

The protein solution was encapsulated in the

mesoporous silica-like material SBA-15 (average pore size 7.2 nm; ACS Material). Two microliter of protein solution (10 mg/mL) was mixed with 1.2 mg of SBA-15, then the sample was suspended in 300 µL of 70 % glycerol and loaded in a 1 mm path-length quartz cuvette (HELLMA) and the far-UV CD spectrum between 195 nm

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Page 22 of 40

and 250 nm recorded on a J-715 spectrometer (JASCO) with a band width of 2.0 nm and the step resolution set at 0.05 nm. Spectra were averaged from 2 scans.

Analytical ultracentrifugation (AUC).

The concentrated protein solution (10

mg/mL) was diluted in the indicated buffer at a concentration of 0.1 mg/mL and loaded into a Beckman AUC sample cell with a 12 mm optical path two-channel centerpiece, with the same buffer in the reference sector. The cell was then loaded into an An60Ti rotor and spun at 60,000 rpm at 20 ℃ and the absorbance at 280 nm recorded at 10 min intervals over a period of 5-6 h.

The partial specific volume ( ),

buffer density, and viscosity were calculated using SEDNTERP software (version 1.09), the sedimentation profile was analyzed using SEDFIT software (version 13.0b), and the sedimentation velocity data were analyzed using the c(s) method of distribution to characterize the sedimentation coefficient distribution of all species in solution.

Transition electron microscopy (TEM).

Protein solution (10 mg/mL) was diluted

to 0.1 mg/mL using the same dissolving buffer, protein samples (10 µL) were loaded on carbon-only coated 300-mesh copper grids and left for 3 min for adsorption to occur, then negative staining was performed for 3 min using freshly filtered 2 %

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uranyl acetate.

After drying, the samples were viewed using a Hitachi H-7000

electron microscope (Hitachi) set at 75 kV.

Electron spin resonance (ESR) spectroscopy.

For continuous wave (cw)-ESR

studies, 20 µL protein solution (10 mg/mL) was mixed with 12 mg of SBA-15, then the sample was directly loaded into a 4 mm outer diameter quartz ESR tube sealed with Parafilm, as previously demonstrated (24).

For DEER-ESR studies, 80 %

glycerol was used as cryoprotectant and was mixed with the protein solution in a 1:1 volume ratio.

DEER was performed at 50 K, following a commonly-used cooling

approach. Briefly, the sample tube was plunge-cooled in liquid nitrogen, then transferred into the ESR probehead, pre-cooled to 50 K using the helium flow system. The sample was then loaded into two 0.8 mm outer diameter capillaries before introducing into a 4 mm outer diameter quartz ESR tube.

ESR spectra were

recorded on a Bruker ELEXSYS E580 CW/Pulse spectrometer (Bruker) equipped with a dielectric resonator (ER4118X-MD5W) and a helium gas flow system (4118CF and 4112HV). Cw-ESR studies were performed at 200 K at an operating frequency of 9.4 GHz (X-band) with 1.5 mW incident microwave power. The swept magnetic range was 200 Gauss.

23



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Supporting Information Available: This material is available free of charge via the Internet at http://pubs.acs.org.

Acknowledgments This work was supported by the Ministry of Science and Technology, Taiwan, R.O.C (grant nos. 102-2113-M-001-013-MY3, 102-2628-M-007-003-MY3, and 103-2627-M-007-005).

We thank Dr. I. Baskakov for kindly providing the plasmid

containing the mouse PrP gene.

The ESI-TOF mass identification of proteins was

performed by the Core Facilities at the Institute of Biological Chemistry, Academia Sinica, supported by the Ministry of Science and Technology and the Academia Sinica. We thank Mr. T.-L. Lin and the Core Facility of the Institute of Cellular and Organismic Biology, Academia Sinica, Taiwan, for assistance with the transmission electron microscopy. We thank Dr. C. Jao of the Biophysics Core Facility of the Academia Sinica and Dr. M.-R. Ho of the Biophysics Instrumentation Laboratory of the Institute of Biological Chemistry, Academia Sinica for performing the analytical ultracentrifugation study.

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References 1.

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DeMarco, M. L., and Daggett, V. (2004) From conversion to aggregation: protofibril formation of the prion protein, Proc. Natl. Acad. Sci. USA 101, 2293-2298. Cobb, N. J., Sonnichsen, F. D., McHaourab, H., and Surewicz, W. K. (2007) Molecular architecture of human prion protein amyloid: a parallel, in-register β-structure, Proc. Natl. Acad. Sci. USA 104, 18946-18951. Lu, X., Wintrode, P. L., and Surewicz, W. K. (2007) β-Sheet core of human prion protein amyloid fibrils as determined by hydrogen/deuterium exchange, Proc. Natl. Acad. Sci. USA 104, 1510-1515. Nazabal, A., Hornemann, S., Aguzzi, A., and Zenobi, R. (2009) Hydrogen/deuterium exchange mass spectrometry identifies two highly protected regions in recombinant full-length prion protein amyloid fibrils, J. Mass Spectrom. 44, 965-977. Jackson, G. S., Hosszu, L. L., Power, A., Hill, A. F., Kenney, J., Saibil, H., Craven, C. J., Waltho, J. P., Clarke, A. R., and Collinge, J. (1999) Reversible

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conversion of monomeric human prion protein between native and fibrilogenic conformations, Science 283, 1935-1937. Bocharova, O. V., Breydo, L., Parfenov, A. S., Salnikov, V. V., and Baskakov, I.

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V. (2005) In vitro conversion of full-length mammalian prion protein produces amyloid form with physical properties of PrPSc, J. Mol. Biol. 346, 645-659. Sang, J. C., Lee, C. Y., Luh, F. Y., Huang, Y. W., Chiang, Y. W., and Chen, R. P. (2012) Slow spontaneous α-to-β structural conversion in a non-denaturing neutral condition reveals the intrinsically disordered property of the

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disulfide-reduced recombinant mouse prion protein, Prion 6, 489-497. Novitskaya, V., Bocharova, O. V., Bronstein, I., and Baskakov, I. V. (2006) Amyloid fibrils of mammalian prion protein are highly toxic to cultured cells and primary neurons, J. Biol. Chem. 281, 13828-13836. Burns, C. S., Aronoff-Spencer, E., Legname, G., Prusiner, S. B., Antholine, W. E., Gerfen, G. J., Peisach, J., and Millhauser, G. L. (2003) Copper coordination in the full-length, recombinant prion protein, Biochemistry 42, 6794-6803. 25



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Inanami, O., Hashida, S., Iizuka, D., Horiuchi, M., Hiraoka, W., Shimoyama, Y., Nakamura, H., Inagaki, F., and Kuwabara, M. (2005) Conformational

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change in full-length mouse prion: a site-directed spin-labeling study, Biochem. Biophys. Res. Commun. 335, 785-792. Watanabe, Y., Inanami, O., Horiuchi, M., Hiraoka, W., Shimoyama, Y., Inagaki, F., and Kuwabara, M. (2006) Identification of pH-sensitive regions in the mouse prion by the cysteine-scanning spin-labeling ESR technique, Biochem. Biophys. Res. Commun. 350, 549-556.

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Watanabe, Y., Hiraoka, W., Shimoyama, Y., Horiuchi, M., Kuwabara, M., and Inanami, O. (2008) Instability of familial spongiform encephalopathy-related prion mutants, Biochem. Biophys. Res. Commun. 366, 244-249.

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Lundberg, K. M., Stenland, C. J., Cohen, F. E., Prusiner, S. B., and Millhauser, G. L. (1997) Kinetics and mechanism of amyloid formation by the prion protein H1 peptide as determined by time-dependent ESR, Chem. Biol. 4, 345-355. Chiang, Y. W., Otoshima, Y., Watanabe, Y., Inanami, O., and Shimoyama, Y.

15.

(2008) Dynamics and local ordering of spin-labeled prion protein: an ESR simulation study of a highly PH-sensitive site, J. Biomol. Struct. Dyn. 26, 355-366. 16.

Baute, D., Frydman, V., Zimmermann, H., Kababya, S., and Goldfarb, D. (2005) Properties of the silica layer during the formation of MCM-41 studied by EPR of a silica-bound spin probe, J. Phys. Chem. B 109, 7807-7816.

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Kuo, Y. H., Tseng, Y. R., and Chiang, Y. W. (2013) Concurrent observation of bulk and protein hydration water by spin-label ESR under nanoconfinement, Langmuir 29, 13865-13872.

18.

Okazaki, M., Anandan, S., Seelan, S., Nishida, M., and Toriyama, K. (2007) Spin-probe ESR study on the entrapment of organic solutes by the nanochannel of MCM-41 in benzene, Langmuir 23, 1215-1222.

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Okazaki, M., Jin, P., Ohta, K., and Toriyama, K. (2009) Transportation of aqueous and alcoholic solutions through the nanochannel of MCM-41: A spin probe electron spin resonance study, J. Phys. Chem. C 113, 11086-11094. Okazaki, M., Lwamoto, S., Sueishi, Y., and Toriyama, K. (2008) The condensation process of alcohol molecules in the nanochannel of MCM-41: A spin-probe ESR study, J. Phys. Chem. C 112, 786-793. Okazaki, M., and Toriyama, K. (2005) Entrapment of organic solutes by the

22.

water cage in the nanochannel of MCM-41, J. Phys. Chem. B 109, 20068-20071. Okazaki, M., and Toriyama, K. (2005) Spin-probe ESR study on the dynamics

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of liquid molecules in the MCM-41 nanochannel: Temperature dependence on 2-propanol and water, J. Phys. Chem. B 109, 13180-13185. 23.

Ruthstein, S., Frydman, V., and Goldfarb, D. (2004) Study of the initial formation stages of the mesoporous material SBA-15 using spin-labeled block co-polymer templates, J. Phys. Chem. B 108, 9016-9022.

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Huang, Y. W., Lai, Y. C., Tsai, C. J., and Chiang, Y. W. (2011) Mesopores provide an amorphous state suitable for studying biomolecular structures at cryogenic temperatures, Proc. Natl. Acad. Sci. USA 108, 14145-14150.

25.

Sung, T. C., and Chiang, Y. W. (2010) Identification of complex dynamic modes on prion protein peptides using multifrequency ESR with mesoporous materials, Phys. Chem. Chem. Phys. 12, 13117-13125.

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Tsai, C. J., and Chiang, Y. W. (2012) Effects of anisotropic nanoconfinement on rotational dynamics of biomolecules: An electron spin resonance study, J. Phys. Chem. C 116, 19798-19806. Huang, Y.-W., and Chiang, Y.-W. (2011) Spin-label ESR with nanochannels to improve the study of backbone dynamics and structural conformations of

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polypeptides, Phys. Chem. Chem. Phys. 13, 17521-17531. Maiti, N. R., and Surewicz, W. K. (2001) The role of disulfide bridge in the folding and stability of the recombinant human prion protein, J. Biol. Chem.

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276, 2427-2431. Chiang, Y. W., Zheng, T. Y., Kao, C. J., and Horng, J. C. (2009) Determination of interspin distance distributions by cw-ESR is a single linear inverse

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problem, Biophys. J. 97, 930-936. Chiang, Y. W., Borbat, P. P., and Freed, J. H. (2005) The determination of pair distance distributions by pulsed ESR using Tikhonov regularization, J. Magn.

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Reson. 172, 279-295. Hosszu, L. L., Trevitt, C. R., Jones, S., Batchelor, M., Scott, D. J., Jackson, G. S., Collinge, J., Waltho, J. P., and Clarke, A. R. (2009) Conformational

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properties of β-PrP, J. Biol. Chem. 284, 21981-21990. Bjorndahl, T. C., Zhou, G. P., Liu, X., Perez-Pineiro, R., Semenchenko, V., Saleem, F., Acharya, S., Bujold, A., Sobsey, C. A., and Wishart, D. S. (2011) Detailed biophysical characterization of the acid-induced PrPC to PrPβ conversion process, Biochemistry 50, 1162-1173.

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Smythe, M. L., Nakaie, C. R., and Marshall, G. R. (1995) α-Helical versus 310-helical conformation of alanine-based peptides in aqueous solution: An electron spin resonance investigation, J. Am. Chem. Soc. 117, 10555-10562. Makarava, N., and Baskakov, I. V. (2008) Expression and purification of full-length recombinant PrP of high purity, Methods Mol. Biol. 459, 131-143. 27



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Page 28 of 40

Abbreviations: PrP, prion protein; mPrPwt, recombinant mouse full-length PrP containing residues 23-230; mPrP-CtoA, a mPrPwt mutant in which C179 and C214 are both replaced by Ala; S132C, D144C, R151C, N174C, N181C, Q212C, and Q217C are mPrP-CtoA mutants in which S132, D144, R151, N174, N181, Q212, or Q217 is replaced by Cys, while S132R1, D144R1, R151R1, N174R1, N181R1, Q212R1, and Q217R1 are S132C, D144C, R151C, N174C, N181C, Q212C, and Q217C with a nitroxide-based MTSSL probe attached to the side-chain of the Cys residue to form an R1 side chain. The numbering of residues is based on the hamster prion sequence in order to be consistent with our previous work.

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Figure legends: Figure 1.

(a) Diagram showing the three α-helices and two β-strands of prion

protein and the mutations constructed for spin-labeling in this study. shown as red cylinders and β-strands as green arrows.

α-helices are

The mPrP-CtoA construct

was generated by replacing the two residues C179 and C214 (the only Cys residues in the protein) of mPrP with Ala.

The positions of the 7 cysteine mutations generated,

S132C, D144C, R151C, N174C, N181C, Q212C, and Q217C, are marked. The numbering of residues is based on the hamster prion sequence in order to be consistent with our previous publications (the mouse PrP sequence numbers are one lower than the hamster ones).

(b) Illustration showing proteins encapsulated in

nanochannels.

Figure 2.

Disulfide-lacking mouse PrP in the α-state has a native-like spatial

contact between S132 and Q217.

To prepare prion protein in the α- and β-states,

S132R1/Q217R1 was dissolved in 0.5 mM NaOAc (pH 5) or 10 mM NaOAc (pH 5) at a final protein concentration of 10 mg/mL.

For CD and ESR studies, it was

encapsulated at this concentration in nanochannels, whereas, for the other studies, it was diluted in the same buffer to 0.1 mg/ml and used as such for AUC and TEM studies.

(a&b) CD spectrum of S132R1/Q217R1 in the α-state (a) or β-state (b)

29



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encapsulated in nanochannels. α-state (c) or β-state (d).

Page 30 of 40

(c&d) AUC spectrum of S132R1/Q217R1 in the

(e&f) TEM image of S132R1/Q217R1 in the α-state (e) or

β-state (f); the bars represent 100 nm in (e) and 500 nm in (f).

(g&h) Cw-ESR

spectrum of S132R1/Q217R1 (red line) in the α-state (g) or β-state (h) in nanochannels compared to the summed spectrum for the two single-labeled mutants (S132R1+Q217R1) (blue line) under the same conditions.

(i) P(r) for

S132R1/Q217R1 in the α-state. (j) Native structure of mouse PrP(121-231) (PDB: 1AG2); residues S132 and Q217 are indicated.

Figure 3.

Structural studies on helix 1 in the α- and β-states.

To prepare the prion

protein in the α- or β-states, D144R1/R151R1 was dissolved, respectively, in 0.5 mM NaOAc (pH 5) or 5 mM NaOAc (pH 7) and used as described in Figure 2.

(a&b)

CD spectrum of D144R1/R151R1 in the α-state (a) or β-state (b) encapsulated in nanochannels. (c&d) AUC spectrum of D144R1/R151R1 in the α-state (c) or β-state (d).

(e&f) TEM image of D144R1/R151R1 in the α-state (e) or β-state (f); the bar

represents 100 nm.

(g&h) Cw-ESR spectrum of D144R1/R151R1 in the α-state (g)

or β-state (h) in nanochannels compared to the summed spectrum for the two single-labeled mutants (D144R1+R151R1) under the same conditions.

(i&j) P(r)

results for the cw-ESR spectrum of D144R1/R151R1 in the α-state (i) or β-state (j).

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Figure 4.

Structural studies of helix 2 in the α- and β-states.


protein in the α- or β-states, N174R1/N181R1 was dissolved, respectively, in 0.5 mM NaOAc (pH 5) or 0.5 mM NaOAc (pH 7) and used as described in Figure 2. (a&b) CD spectrum of N174R1/N181R1 in the α-state (a) or β-state (b) in nanochannels. (c&d) AUC spectrum of N174R1/N181R1 in the α-state (c) or β-state (d).

(e&f)

TEM image of N174R1/N181R1 in the α-state (e) or β-state (f); the bar represents 100 nm.

(g&h) Cw-ESR spectrum of N174R1/N181R1 in the α-state (g) or β-state

(h) in nanochannels compared to the summed spectrum for the two single-labeled mutants (N174R1+N181R1) under the same conditions.

(i) P(r) of N174R1/N181R1

in the α-state. (j) Time-domain data for the DEER study on N174R1/N181R1 in the β-state (black line); the red trace corresponds to the simulated fit using the P(r); the distance distribution P(r) is shown in the inset.

Figure 5.

Structural studies of helix 3 in the α- and β-states.


protein in the α- or β-state, Q212R1/Q217R1 was dissolved in, respectively, 0.5 mM NaOAc (pH 5) or 10 mM NaOAc (pH 7) and used as described in Figure 2.

(a&b)

CD spectrum of Q212R1/Q217R1 in the α-state (a) or β-state (b) in nanochannels. (c&d) AUC spectrum of Q212R1/Q217R1 in the α-state (c) or β-state (d).

(e&f)

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TEM image of Q212R1/Q217R1 in the α-state (e) or β-state (f); the bars represent 100 nm.

(g&h) Cw-ESR spectrum of Q212R1/Q217R1 in the α-state (g) or β-state

(h) in nanochannels compared to the summed spectrum for the single-labeled mutants (Q212R1+Q217R1) under the same conditions.

(i&j) P(r) result for the cw-ESR

spectrum of Q212R1/Q217R1 in the α-state (i) or β-state (j).

Figure 6.

Spontaneous α-to-β structural conversion of S132R1/Q217R1 and

N174R1/N181R1

during

incubation

in

solution

or

in

nanochannels.

S132R1/Q217R1 and N174R1/N181R1 in the α-state were prepared by dissolving the proteins in 0.5 mM NaOAc (pH 5) at a concentration of 10 mg/mL.

(a&b) CD

spectra of S132R1/Q217R1 (a) and N174R1/N181R1 (b) in the α-state recorded without incubation (0 day) or after incubation at 4 ℃ for the indicated time, followed by encapsulation.

(c&d) Normalized cw-ESR spectra of S132R1/Q217R1

(c) and N174R1/N181R1 (d) originally in the α-state recorded after incubation inside nanochannels for the indicated time. The cw-ESR spectra of S132R1/Q217R1 and N174R1/N181R1 in the β-state were taken from Figs. 2h and 4h.

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TOC

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Figure 1. (a) Diagram showing the three α-helices and two β-strands of prion protein and the mutations constructed for spin-labeling in this study. α-helices are shown as red cylinders and β-strands as green arrows. The mPrP-CtoA construct was generated by replacing the two residues C179 and C214 (the only Cys residues in the protein) of mPrP with Ala. The positions of the 7 cysteine mutations generated, S132C, D144C, R151C, N174C, N181C, Q212C, and Q217C, are marked. The numbering of residues is based on the hamster prion sequence in order to be consistent with our previous publications (the mouse PrP sequence numbers are one lower than the hamster ones). (b) Illustration showing proteins encapsulated in nanochannels. 254x190mm (96 x 96 DPI)


Page 34 of 40

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Figure 2. Disulfide-lacking mouse PrP in the α-state has a native-like spatial contact between S132 and Q217. To prepare prion protein in the α- and β-states, S132R1/Q217R1 was dissolved in 0.5 mM NaOAc (pH 5) or 10 mM NaOAc (pH 5) at a final protein concentration of 10 mg/mL. For CD and ESR studies, it was encapsulated at this concentration in nanochannels, whereas, for the other studies, it was diluted in the same buffer to 0.1 mg/ml and used as such for AUC and TEM studies. (a&b) CD spectrum of S132R1/Q217R1 in the α-state (a) or β-state (b) encapsulated in nanochannels. (c&d) AUC spectrum of S132R1/Q217R1 in the α-state (c) or β-state (d). (e&f) TEM image of S132R1/Q217R1 in the α-state (e) or β-state (f); the bars represent 100 nm in (e) and 500 nm in (f). (g&h) Cw-ESR spectrum of S132R1/Q217R1 (red line) in the α-state (g) or β-state (h) in nanochannels compared to the summed spectrum for the two single-labeled mutants (S132R1+Q217R1) (blue line) under the same conditions. (i) P(r) for S132R1/Q217R1 in the α-state. (j) Native structure of mouse PrP(121-231) (PDB: 1AG2); residues S132 and Q217 are indicated. 66x119mm (300 x 300 DPI)



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 ACS Paragon Plus Environment

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Figure 3. Structural studies on helix 1 in the α- and β-states. To prepare the prion protein in the α- or βstates, D144R1/R151R1 was dissolved, respectively, in 0.5 mM NaOAc (pH 5) or 5 mM NaOAc (pH 7) and used as described in Figure 2. (a&b) CD spectrum of D144R1/R151R1 in the α-state (a) or β-state (b) encapsulated in nanochannels. (c&d) AUC spectrum of D144R1/R151R1 in the α-state (c) or β-state (d). (e&f) TEM image of D144R1/R151R1 in the α-state (e) or β-state (f); the bar represents 100 nm. (g&h) Cw-ESR spectrum of D144R1/R151R1 in the α-state (g) or β-state (h) in nanochannels compared to the summed spectrum for the two single-labeled mutants (D144R1+R151R1) under the same conditions. (i&j) P(r) results for the cw-ESR spectrum of D144R1/R151R1 in the α-state (i) or β-state (j). 66x112mm (300 x 300 DPI)



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Figure 4. Structural studies of helix 2 in the α- and β-states. To prepare the prion protein in the α- or βstates, N174R1/N181R1 was dissolved, respectively, in 0.5 mM NaOAc (pH 5) or 0.5 mM NaOAc (pH 7) and used as described in Figure 2. (a&b) CD spectrum of N174R1/N181R1 in the α-state (a) or β-state (b) in nanochannels. (c&d) AUC spectrum of N174R1/N181R1 in the α-state (c) or β-state (d). (e&f) TEM image of N174R1/N181R1 in the α-state (e) or β-state (f); the bar represents 100 nm. (g&h) Cw-ESR spectrum of N174R1/N181R1 in the α-state (g) or β-state (h) in nanochannels compared to the summed spectrum for the two single-labeled mutants (N174R1+N181R1) under the same conditions. (i) P(r) of N174R1/N181R1 in the α-state. (j) Time-domain data for the DEER study on N174R1/N181R1 in the β-state (black line); the red trace corresponds to the simulated fit using the P(r); the distance distribution P(r) is shown in the inset. 66x113mm (300 x 300 DPI)


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Figure 5. Structural studies of helix 3 in the α- and β-states. To prepare the prion protein in the α- or βstate, Q212R1/Q217R1 was dissolved in, respectively, 0.5 mM NaOAc (pH 5) or 10 mM NaOAc (pH 7) and used as described in Figure 2. (a&b) CD spectrum of Q212R1/Q217R1 in the α-state (a) or β-state (b) in nanochannels. (c&d) AUC spectrum of Q212R1/Q217R1 in the α-state (c) or β-state (d). (e&f) TEM image of Q212R1/Q217R1 in the α-state (e) or β-state (f); the bars represent 100 nm. (g&h) Cw-ESR spectrum of Q212R1/Q217R1 in the α-state (g) or β-state (h) in nanochannels compared to the summed spectrum for the single-labeled mutants (Q212R1+Q217R1) under the same conditions. (i&j) P(r) result for the cw-ESR spectrum of Q212R1/Q217R1 in the α-state (i) or β-state (j). 66x112mm (300 x 300 DPI)



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Figure 6. Spontaneous α-to-β structural conversion of S132R1/Q217R1 and N174R1/N181R1 during incubation in solution or in nanochannels. S132R1/Q217R1 and N174R1/N181R1 in the α-state were prepared by dissolving the proteins in 0.5 mM NaOAc (pH 5) at a concentration of 10 mg/mL. (a&b) CD spectra of S132R1/Q217R1 (a) and N174R1/N181R1 (b) in the α-state recorded without incubation (0 day) or after incubation at 4 ℃ for the indicated time, followed by encapsulation. (c&d) Normalized cw-ESR spectra of S132R1/Q217R1 (c) and N174R1/N181R1 (d) originally in the α-state recorded after incubation inside nanochannels for the indicated time. The cw-ESR spectra of S132R1/Q217R1 and N174R1/N181R1 in the β-state were taken from Figs. 2h and 4h. 66x85mm (300 x 300 DPI)


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Mechanism of the α-to-β structural conversion of mouse prion protein is studied by Rita Chen’s group. Double spin-labeled prion protein is encapsulated in naochannels and the spin-spin distance is measured by ESR spectroscopy. 214x279mm (300 x 300 DPI)


Revealing Structural Changes of Prion Protein during Conversion from

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